A biosensor is a sensor which applies a biological material as a molecule identification element by utilizing a molecule identification function of the biological material such as a microorganism, an enzyme, an antibody, a DNA, and an RNA to determine the content of substrate in an analyte. That is, the biosensor determines the content of the substrate contained in the analyte with utilizing a reaction which occurs when the biological material identifies the target substrate, such as oxygen consumption caused by a respiration of a microorganism, an enzyme reaction, light emission and the like.
Among various biosensors described above, practical use of an enzyme sensor which is a biosensor for such as glucose, lactic acid, cholesterol, and amino acid is advancing, and the enzyme biosensor is utilized for medical measurement and food industry. The enzyme sensor reduces electron transporters by electrons which are generated by reactions between the substrate (for example, glucose) contained in the analyte (for example, sample solution such as blood) and the enzyme or the like, and the measurement apparatus for the enzyme sensor electrochemically measures an amount of reduction of the electron transporters, thereby quantitatively analizing the content of the substrate in the analyte.
As described above, the determination of the substrate contained in body fluid of a human body is very important for a diagnosis or a therapy for a specific physiological abnormality. Specifically, it is necessary for a diabetic to frequently comprehend the concentration of glucose in the blood.
Conventionally, various types of the biosensors are proposed. Hereinafter, a prior art biosensor will be described with reference to FIG. 1. FIG. 1(a) is an exploded perspective view illustrating a construction of a biosensor, and FIG. 1(b) is a plan view of the biosensor shown in FIG. 1(a).
In FIG. 1(a), a biosensor 15 comprises an insulating substrate 1 made of polyethylene terephthalate and the like (hereinafter, merely referred to as “a substrate”), a spacer 6 having a notch 7, an insulating substrate 8 provided with an air hole 9, and a reagent layer 5, which are integrally positioned with the spacer 6 and the reagent layer 5 being sandwiched between the insulating substrate 8 and the substrate 1.
A conductive layer 10 made of an electrically conductive material such as precious metal like gold or palladium or carbon is formed on the surface of the substrate 1 by using a screen printing method or a sputtering vapor deposition method, and the conductive layer 10 on the substrate 1 is divided by plural slits to produce a counter electrode 3, a measuring electrode 2 and a detection electrode 4. Then, substantially arc-shaped slits 13 and 14 are formed on the counter electrode 3. While FIG. 1 shows that the conductive layer 10 is provided on the whole surface of the substrate 1, the conductive layer 10 may be formed on a part of the substrate 1, and the respective electrodes 2, 3, and 4 may be formed on parts of the substrate 1.
The spacer 6 is positioned so as to cover the counter electrode 3, the measuring electrode 2 and the detection electrode 4 on the substrate 1. Then, an analyte supply path 7a is produced by the rectangular notch 7 provided at the center of the front edge of the spacer 6. When a sample solution such as blood which is an analyte is spotted onto an analyte spot portion 15a which is positioned at an end of the analyte supply path 7a, the sample solution is substantially horizontally sucked toward the air hole 9 due to a capillary phenomenon.
The reagent layer 5 is produced by applying a reagent containing an enzyme, an electron acceptor, a hydrophilic high polymer and the like onto the counter electrode 3, the measuring electrode 2, and the detection electrode 4 on the substrate 1, which are exposed from the notch 7 of the spacer 6, and a spread of the applied reagent on the substrate 1 is controlled by the arc-shaped slits 13 and 14 which are formed on the counter electrode 3.
Here, as an enzyme to be contained in the reagent, glucose oxidase, lactate oxidase, cholesterol oxidase, cholesterol esterase, uricase, ascorbic acid oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase and the like can be used. Further, while potassium ferricyanide is preferably used as the electron accepter, p-benzoquinone and a derivative thereof, phenazine methyl sulfate, methylene blue, ferrocene and a derivative thereof, and the like, may be used other than potassium ferricyanide. The specific examples of the enzyme and the electron accepter contained in the reagent layer 5 of the biosensor 15, which are cited here, are particularly suitable for determining a content of glucose, lactic acid, and cholesterol which are substrates contained in blood of a human body as an analyte. When, for example, a glucose in blood of a human body is determined using this biosensor 15, glucose dehydrogenase and potassium ferricyanide are used as an oxidation-reduction enzyme and an electron accepter which are to be contained in the reagent layer 5, respectively.
Hereinafter, a case where the content of a substrate in an analyte is determined using the biosensor 15 having the above-described construction will be described. While a case where glucose contained in blood of a human body is determined using the biosensor 15 is described, lactic acid, cholesterol, or other substrates can also be determined with appropriately selecting an enzyme contained in the reagent layer 5 of the biosensor 15.
Initially, when a blood taken from a human body is spotted onto the analyte spot portion 15a of the analyte supply path 7a of the biosensor 15, the oxidation-reduction enzyme, i.e., glucose dehydrogenase and the electron accepter, i.e., potassium ferricyanide which are contained in the reagent layer 5 are dissolved into the blood which is sucked in the analyte supply path 7a, and thereby an enzyme reaction proceeds between the glucose which is a substrate in the blood and the oxidation-reduction enzyme, and further, the enzyme reaction makes the potassium ferricyanide serving as an electron accepter reduced thereby to generate ferrocyanide (potassium ferrocyanide). Then, a series of these reactions (the enzyme reaction of the oxidation-reduction enzyme, and the reduction of the electron accepter) mainly proceed in the analyte supply path 7a. 
Then, the above-described reduced potassium ferrocyanide which was serving as an electron accepter is electrochemically oxidized and a current value which is obtained this time with associated with the above-described electrochemical change is read by the counter electrode 3, the measuring electrode 2, and the detection electrode 4 on the conductive layer 10 in a measurement apparatus for a biosensor, which will be described later, and thereby the concentration of glucose in the blood is measured on the basis of the current value.
Then, the determination of the substrate in the analyte liquid is performed by inserting the biosensor 15 into the measurement apparatus 16 for the biosensor as shown in FIG. 2.
Hereinafter, an operation for determining glucose in blood of a human body with a biosensor system comprising the biosensor 15 and the measurement apparatus 16 for biosensor will be described with reference to FIG. 2. FIG. 2 is a diagram illustrating a construction of a prior art biosensor system.
Initially, the construction of the biosensor will be described. As shown in FIG. 2, the biosensor system comprises the above-described biosensor 15 and the measurement apparatus 16 for the biosensor to which the biosensor 15 is detachably mounted, and it determines an amount of substrate contained in an analyte spotted onto the analyte spot portion 15a of the biosensor 15 with the measurement apparatus 16. Then, the measurement apparatus 16 for biosensor comprises an insertion portion 17 to which the biosensor 15 is detachably mounted, a driving power supply (not shown) for applying a voltage to the electrodes of the biosensor, and a display unit 18 which displays the result of determining the substrate in the analyte, which is obtained by applying the voltage by the driving power supply. Then, the measurement apparatus 16 for the biosensor and the respective electrodes of the biosensor 15 may be connected through lead wires.
In a case where the content of substrate such as glucose in blood is determined using a biosensor of such construction, a user initially inserts the biosensor 15 into the measurement apparatus 16. Then, the user spots blood onto the analyte spot portion 15a in a state where a constant voltage is applied between the counter electrode 3 and the measuring electrode 2 on the substrate 1 of the biosensor 15 in the measurement apparatus 16. The spotted blood is sucked into the inside of the biosensor 15 toward the air hole 9, and thereby the reagent layer 5 starts to be dissolved.
At this time, the measurement apparatus 16 detects an electrical change which occurs between the electrodes 2 and 3 in the biosensor 15, to start the determination operation.
In the measurement apparatus 16 for the biosensor, a constant voltage is applied between the electrodes 2 and 3 in the biosensor 15 which is inserted into the measurement apparatus 16, and it is detected that a sample solution which is blood is spotted onto the analyte spot portion 15a of the analyte supply path 7a, which is an enzyme reaction layer of the biosensor 15. Then, application of a voltage between the electrodes 2 and 3 is temporarily halted, and a constant voltage is again applied after a given time period, and then a current which flows between the electrodes 2 and 3 is measured, and thereby glucose in the blood is determined and the blood sugar level is calculated (for example, refer to claim 2 of the Japanese Published Patent Application No. Hei. 3-287064).
As a performance which is recently requested for such a biosensor system, further shortening of the measurement time is raised.
When determination of substrate in blood is performed by the above-described biosensor at high speed, the viscosity of the blood as an analyte affects substantial influences upon the precision of measurement. As a determination method which can solve these problems and enables performing a high precision measurement with that biosensor, a method is disclosed in which the measurement apparatus 16 applies a first potential between the counter electrode 3 and the measuring electrode 2 in the biosensor for a first time period, and then application of the potential is halted for a waiting period, and a second potential which is lower than the first potential is applied between the counter electrode 3 and the measuring electrode 2 for a second time period after the passage of the waiting period, and thereby an output current is measured (for example, refer to claim 21 of the International Application Publication No. WO02/44705A1).
Recently, a commodity product having various different functions is developed as a compact and simple blood sugar level measurement system for determining the blood sugar level. For example, an importance is placed especially upon a field of data management such as management and processing of the measurement data in the blood sugar level measurement system. Generally, for the biosensor system comprising the above-described biosensor and measurement apparatus for the biosensor, the administration of the precision of measurement is periodically performed using its dedicated standard solution so as to maintain and control the precision of measurement.
Here, as the conventional standard solution for the biosensor, one containing water, a predetermined amount of glucose, xanthan, and phosphate as a reaction rate modifier is disclosed (for example, refer to claim 1 of the International Application Publication No. WO93/21928-A). Further, as another example, serum-free control reagent containing a mixture of a predetermined amount of glucose used for measuring the glucose, water, a thickener, a buffer, a preservative, a surface-active agent, a coloring or color-forming compound, and the like are disclosed (for example, refer to claims 1 to 8 of the International Application Publication No. WO95/13536-A).
Then, in the prior art biosensor system which controls the precision of measurement of the measurement apparatus using the standard solution, in order to prevent the measurement data of the standard solution from being confused in being processed with the measurement data of the body fluid and the like used as a normal sample solution, the measurement apparatus is previously switched to a measurement mode for the standard solution with a predetermined manual operation when the standard solution is introduced into the biosensor system, and thereby the measurement data of the standard solution and the measurement data of the sample solution are distinguished from each other.
On the other hand, in recent years, a means for automatically identifying the kind of analyte liquid is provided. Disclosed as that method is one in which a ratio between a current value which is measured for each sample solution that is an analyte and a value obtained by time-differentiating the current value is set as a discrimination parameter for discriminating between the respective analyte liquids, and a discrimination function employing the discrimination parameter as an independent variable is defined for discriminating among plural kinds of target analyte liquids, and a numeric value which is obtained by substituting the value of the discrimination parameter into the discrimination function is taken as a discrimination index, and thereby the kinds of samples are automatically discriminated on the basis of the discrimination index. (For example, refer to claim 1 of International Application Publication No. WO01/40787A1).
However, the above-described method for automatically identifying the kind of analyte liquid cannot accomplish the identification of the kind of analyte liquid with high precision due to the following various factors, and the method is not currently put into practical use.
Hereinafter, the factors will be specifically described with reference to FIG. 3. FIG. 3 is a diagram illustrating waveforms of oxidation current values which are measured when a voltage is applied to the biosensor onto which a conventional standard solution or bloods under various conditions are spotted in the biosensor system. FIG. 3(a) shows current waveforms of a conventional standard solution a and three kinds of bloods b to d having different hematocrit values from each other, FIG. 3(b) shows current waveforms of the conventional sample solution a and three kinds of bloods e to f containing different interfering substances from each other, and FIG. 3(c) shows current waveforms of the conventional standard solution a and three kinds of bloods h to j having different environmental temperatures from each other. Then, the measurement shown in FIG. 3 is performed on the basis of a measurement profile shown in FIG. 4. More specifically, as indicated in the above-described International Application Publication No. WO02/44705A1, the measurement apparatus 16 applies a first potential between the counter electrode 3 and the measuring electrode 2 of the biosensor onto which the respective analytes are spotted for a first potential time period (from time t0 to t1), and thereafter stops the application for a waiting period (from time t1 to t2), and applies a second potential which is lower than the first potential for a second potential time period (from time t2 to t3) after the waiting period has passed. Here, the measurement is performed under conditions where the first potential is 0.5V and the second potential is 0.2V, and the first potential time period is 6 seconds, the waiting period is 6 seconds, and the second potential time period is 3 seconds. However, it is required to appropriately set the applied potential and the application time period in accordance with the electrode materials used for the biosensor 15 as well as conditions of the reagent layer 5.
Initially, as a first factor, personal differences in the constituent components of bloods which are used as sample solutions are raised.
Since the hematocrit values which exert influences onto the viscosities of the bloods vary among users, the current waveforms of bloods having different hematocrit values from each other show various shapes as shown by the waveforms b to d in FIG. 3(a). Then, in FIG. 3(a), “a” denotes a current waveform of a conventional sample solution, “b” denotes a current waveform of a blood having hematocrit value of 20%, “c” denotes a current waveform of a blood having hematocrit value of 45%, and “d” denotes a current waveform of a blood having hematocrit value of 60%.
Further, since various substances called interfering substances which exert influences onto the measurement values, such as ascorbic acid, uric acid, and bilirubin, vary among the users, the current waveforms of bloods containing the above-described interfering substances show various shapes as shown by the waveforms e to g in FIG. 3(b). Then, in FIG. 3(b), “e” denotes a current waveform of a blood containing ascorbic acid (10 mg/dl), “f” denotes a current waveform of a blood containing uric acid (10 mg/dl), and “g” denotes a current waveform of a blood containing bilirubin (10 mg/dl).
As a result, as is apparent from FIGS. 3(a) and 3(b), there is little difference between the current waveform a of the conventional standard solution and the current waveforms b to g of the bloods which vary among individuals as described above, which makes it difficult to discriminate between the conventional standard solution and the respective sample solutions with high precision.
Secondly, as a second factor, environmental conditions under which the users use the blood sugar measurement system are different from each other.
The blood sugar measurement system is used under environmental conditions which would vary among the users. In a case where the environmental conditions where it is used, for example, environmental temperatures, are wide-ranging (for example, 10° C. to 40° C.), the solubility of the substrate contained in the analyte liquid and the reagent layer as well as the reaction rate thereof vary depending on the environmental temperatures, and therefore the current waveforms of bloods having different environmental conditions from each other show various shapes as shown by the waveforms h to j in FIG. 3(c). In FIG. 3(c), “h” denotes a current waveform of a blood having environmental temperature of 40° C., “i” denotes a current waveform of a blood having environmental temperature of 25° C., and “j” denotes a current waveform of a blood having environmental temperature of 10° C. As with the above description, also in this case, as is apparent from FIG. 3(c), there is little difference between the current waveform a of the conventional standard solution and the current waveforms h to j of the bloods having different environmental conditions from each other, which makes it difficult to discriminate between the conventional standard solution and the respective sample solutions with high precision. Then, the environmental temperature of the conventional standard solution a shown in FIG. 3(c) is 25° C.
As described above, the current waveform of the conventional standard solution is very similar to the current waveforms of the bloods as sample solution under various conditions. Therefore, in a case where the measurement apparatus 16 automatically discriminates whether the kind of analyte liquid being measured is a standard solution or a sample solution, on the basis of the current waveforms obtained from the measurement, an error in identifying the kind of analyte liquid is likely to occur. Therefore, the prior art measurement apparatus 16 must be constructed so that it may be switched to a measurement mode with manual operation, as described above.
The present invention is made in view of the above-described problems, and has its object to provide standard solution for a biosensor, which has high precision and can eliminate errors in identifying the kind of analyte liquid, and a determination method for automatically identifying the kind of analyte liquid using the standard solution.